靶向烧伤创面的荧光显像工具:低pH插入肽的应用研究

A fluorescence imaging tool targeting burn wounds:research on the application of pH low insertion peptide

目的利用低pH插入肽(pHLIP)-变体7(var7)-异硫氰酸荧光素(FITC)探索一种靶向烧伤创面的精确显像工具,以更好地进行烧伤清创。方法建立大鼠烧伤模型(n=12),尾静脉注射不同浓度(0.5、1.5和2.0 mg/ml)pHLIP-var7-FITC进行活体荧光显像。通过荧光偶联物聚集至烧伤创面的集中位置,结合病理切片判断创面损伤坏死范围;并检测pHLIP-var7-FITC在心、肝、肾、脑等重要器官的残留及毒性。采用Kruskal-Wallis秩和检验、Bonferroni校正法及单因素方差分析进行数据分析。结果24 h内,0.5 mg/ml组、1.5 mg/ml组、2.0 mg/ml组烧伤创面单位面积荧光光子数分别为1.49(1.31,1.65)、2.46(1.88,2.68)、2.77(1.94,3.10)×107 p·s-1·cm-2·Sr-1,其荧光光子数总体分布差异有统计学意义(H=73.55,P<0.001),浓度较高组荧光强度较强,但1.5 mg/ml组与2.0 mg/ml组荧光光子数差异无统计学意义(P=0.263,Bonferroni校正法)。14个时间节点(0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、5.0、6.0、7.0、8.0、12、24 h)的荧光光子数总体均数差异无统计学意义(F=1.04,P=0.419),组织切片中可见烧伤坏死的组织与荧光显像区域高度一致。心、肝、肾、脑切片未见明显荧光残留。结论在浅Ⅱ度烧伤组织中,24 h内pHLIP-var7-FITC能精准靶向聚集在烧伤创面,呈现烧伤组织与正常组织的清晰界限,辅助临床外科手术清创判断损伤范围。

Objective pH low insertion peptide(pHLIP)-variant 7(var7)-fluorescein isothiocyanate(FITC)was used to explore an accurate imaging tool that targeted burn wounds to better perform burn debridement.Methods Twelve rat models of burn wound were established and pHLIP-var7-FITC with different concentrations(0.5,1.5 and 2.0 mg/ml)were injected from the rat tail vein for in vivo fluorescence imaging.By determining the concentration of fluorescent conjugates to the burn wound,the scope of wound injury necrosis was judged by combining pathological sections,and its residue and toxicity in important organs such as heart,liver,kidneys,and brain were detected.The Kruskal-Wallis rank sum test,Bonferroni correction method and one-way analysis of variance were used for data analysis.Results Within 24 h,the fluorescence photons per unit area of the burn wound in the group of 0.5 mg/ml,1.5 mg/ml and 2.0 mg/ml were 1.49(1.31,1.65),2.46(1.88,2.68),2.77(1.94,3.10)×107 p·s-1·cm-2·Sr-1,with significant differences in the overall distribution of fluorescence photons(H=73.55,P<0.001).The fluorescence intensity was stronger in the group with higher concentration,but with no significant difference in the number of fluorescence photons between the group of 1.5 mg/ml and 2.0 mg/ml(P=0.263,Bonferroni correction method).At 14 time points(0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,5.0,6.0,7.0,8.0,12,24 h),there was no significant difference in the overall mean of fluorescence photons(F=1.04,P=0.419),and the tissue with burn necrosis seen in tissue sections was highly consistent with the fluorescence imaging region.There was no obvious fluorescence residue in the heart,liver,kidney and brain sections.Conclusion In superficial second-degree burn tissue,pHLIP-var7-FITC can accurately target and gather on the burn wound within 24 h,showing a clear boundary between burn tissue and normal tissue,which can assist clinical surgical debridement to determine the extent of injury.